Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-α and -β

Abstract

Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (α1, α2 and β) that are encoded by two separate genes termed ck-α and ck-β. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-α1 and CK-β tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424–K430 for CK-α1 and Q379–K385 for CK-β) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-α1 was found to be essential for α1/α1 homo-dimerization but not for α1/β hetero-dimerization. A mutation of the corresponding D276 of CK-β to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.